Platelet-poor plasma of athletes is a potent inducer of myogenic differentiation of C2C12 myoblasts

Introduction. Blood products, i.e. platelet rich plasma (PRP), leukocyte-poor plasma (PRP) and platelet poor plasma (PPP), have previously been used to improve muscle regeneration. In this study, six months? frozen-stored PPP of individuals who practiced different types of physical exercise was analysed; it could steer mouse C2C12 myoblast cells towards proliferation, migration and myogenic differentiation, and it could affect the morphology/shape of myotubes. Materials and Methods. PPP of male Olympic weightlifters, football players and professional folk dancers, aged 15-19, was collected 12 h post-training and stored for 6 months at -20?C. C2C12 cell proliferation was assessed by MTT test, motility by scratch assay, myogenic differentiation by myotube formation and gelatinase activity by gel-zymography. Results and Conclusions. PPP induced proliferation and migration of C2C12 cells. Proliferative capacity was as follows: weightlifters > dancers > football players; mean migratory capacity was: weightlifters = dancers > football players. PPP induced formation of myotubes; significant inter-individual variations were detected: PPP from weightlifters induced formation of round myotubes, and PPP from football players and dancers induced formation of elongated myotubes. The mean myotube area was as follows: football players > dancers > weightlifters. PPP gelatinolytic activity was observed; it was negatively correlated with C2C12 myoblast proliferation. These results provide general but distinct evidence that PPP of individuals practicing certain types of exercise can specifically modify myoblast morphology/function. This is significant for explaining physiological responses and adaptations to exercise. In conclusion, long-term, frozen-stored PPP preserves its potential to modify myoblast morphology and function.

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2-D08 treatment regulates C2C12 myoblast proliferation and differentiation via the Erk1/2 and proteasome signaling pathways

Journal of Muscle Research and Cell Motility ◽

10.1007/s10974-021-09605-x ◽

2021 ◽

Author(s):

Hyunju Liu ◽

Su-Mi Lee ◽

Hosouk Joung

Keyword(s):

Myogenic Differentiation ◽

C2c12 Cell ◽

C2c12 Cells ◽

Covalent Attachment ◽

Myoblast Differentiation ◽

C2c12 Myoblast ◽

Long Term Effects ◽

Proliferation And Differentiation ◽

Myoblast Proliferation ◽

Myoblast Cells

AbstractSUMOylation is one of the post-translational modifications that involves the covalent attachment of the small ubiquitin-like modifier (SUMO) to the substrate. SUMOylation regulates multiple biological processes, including myoblast proliferation, differentiation, and apoptosis. 2-D08 is a synthetically available flavone, which acts as a potent cell-permeable SUMOylation inhibitor. Its mechanism of action involves preventing the transfer of SUMO from the E2 thioester to the substrate without influencing SUMO-activating enzyme E1 (SAE-1/2) or E2 Ubc9-SUMO thioester formation. However, both the effects and mechanisms of 2-D08 on C2C12 myoblast cells remain unclear. In the present study, we found that treatment with 2-D08 inhibits C2C12 cell proliferation and differentiation. We confirmed that 2-D08 significantly hampers the viability of C2C12 cells. Additionally, it inhibited myogenic differentiation, decreasing myosin heavy chain (MHC), MyoD, and myogenin expression. Furthermore, we confirmed that 2-D08-mediated anti-myogenic effects impair myoblast differentiation and myotube formation, reducing the number of MHC-positive C2C12 cells. In addition, we found that 2-D08 induces the activation of ErK1/2 and the degradation of MyoD and myogenin in C2C12 cells. Taken together, these results indicated that 2-D08 treatment results in the deregulated proliferation and differentiation of myoblasts. However, further research is needed to investigate the long-term effects of 2-D08 on skeletal muscles.

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Doxycycline Inhibits IL-17-Stimulated MMP-9 Expression by Downregulating ERK1/2 Activation: Implications in Myogenic Differentiation

Mediators of Inflammation ◽

10.1155/2016/2939658 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Hristina Obradović ◽

Jelena Krstić ◽

Tamara Kukolj ◽

Drenka Trivanović ◽

Ivana Okić Đorđević ◽

...

Keyword(s):

Inflammatory Diseases ◽

Myogenic Differentiation ◽

C2c12 Cell ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

Interleukin 17 ◽

Myoblast Cells ◽

Pathological Consequence ◽

Muscle Remodeling

Interleukin 17 (IL-17) is a cytokine with pleiotropic effects associated with several inflammatory diseases. Although elevated levels of IL-17 have been described in inflammatory myopathies, its role in muscle remodeling and regeneration is still unknown. Excessive extracellular matrix degradation in skeletal muscle is an important pathological consequence of many diseases involving muscle wasting. In this study, the role of IL-17 on the expression of matrix metalloproteinase- (MMP-) 9 in myoblast cells was investigated. The expression of MMP-9 after IL-17 treatment was analyzed in mouse myoblasts C2C12 cell line. The increase in MMP-9 production by IL-17 was concomitant with its capacity to inhibit myogenic differentiation of C2C12 cells. Doxycycline (Doxy) treatment protected the myogenic capacity of myoblasts from IL-17 inhibition and, moreover, increased myotubes hypertrophy. Doxy blocked the capacity of IL-17 to stimulate MMP-9 production by regulating IL-17-induced ERK1/2 MAPK activation. Our results imply that MMP-9 mediates IL-17’s capacity to inhibit myoblast differentiation during inflammatory diseases and indicate that Doxy can modulate myoblast response to inflammatory induction by IL-17.

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Osteocalcin Induces Proliferation via Positive Activation of the PI3K/Akt, P38 MAPK Pathways and Promotes Differentiation Through Activation of the GPRC6A-ERK1/2 Pathway in C2C12 Myoblast Cells

Cellular Physiology and Biochemistry ◽

10.1159/000481752 ◽

2017 ◽

Vol 43 (3) ◽

pp. 1100-1112 ◽

Cited By ~ 22

Author(s):

Suifeng Liu ◽

Feng Gao ◽

Lei Wen ◽

Min Ouyang ◽

Yi Wang ◽

...

Keyword(s):

Cell Proliferation ◽

P38 Mapk ◽

C2c12 Cell ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

C2c12 Myoblast ◽

C2c12 Myoblasts ◽

Mapk Phosphorylation ◽

Mapk Pathways ◽

Myoblast Cells

Background/Aims: Sarcopenia is characterized by an age-related decline in skeletal muscle plus low muscle strength and/or physical performance. Despite the clinical significance of sarcopenia, the molecular pathways underlying sarcopenia remain elusive. The recent demonstration that undercarboxylated osteocalcin (ucOC) favours muscle function related to insulin sensitivity and glucose metabolism raises the question of whether this hormone may also regulate muscle mass. The present study explored the promotive effects of ucOC in proliferation and differentiation processes of C2C12 myoblasts as well as the possible signalling pathways involved. Methods: The effects of exogenous ucOC on C2C12 myoblasts proliferation were assessed using CCK8 and immunohistological staining assays. C2C12 cells were pretreated with PI3K/Akt or P38 MAPK inhibitors to investigate the possible involvement of the PI3K/Akt and P38 MAPK pathways in proliferation. The levels of Akt, phosphorylated-Akt (p-Akt), P38, and phosphorylated-P38 (p-P38) were measured by Western Blotting. The effects of ucOC on myoblast differentiation were quantified by morphological analysis. A silencing experiment was conducted in which the expression of GPRC6A in C2C12 myoblasts was modified. The expression of GPRC6A, myosin heavy chain (MyHC) and the related ERK1/2 signalling pathway in C2C12 myoblasts were monitored by qRT-PCR and Western Blotting. Results: We showed that treatment with exogenous ucOC stimulated the priming of C2C12 myoblasts proliferation. Inhibition of Akt phosphorylation by wortmannin or inhibition of P38 MAPK phosphorylation by SB203580 decreased C2C12 cell proliferation. Wortmannin also reduced P38 MAPK phosphorylation, whereas SB203580 did not affect Akt activation. Furthermore, ucOC promoted C2C12 myoblast differentiation. Inhibition of ERK1/2 phosphorylation with U0126 decreased C2C12 cell differentiation. Finally, GPRC6A expression was substantially increased after ucOC treatment of C2C12 cells. GPRC6A silencing inhibited Akt, P38 MAPK phosphorylation in C2C12 cells, and ERK1/2 phosphorylation in C2C12 myotubes; GPRC6A silencing also decreased cell proliferation, decreased cell differentiation, and downregulated MyHC expression. Conclusions: The present data suggest that ucOC induces myoblast proliferation via sequential activation of the PI3K/Akt and p38 MAPK pathways in C2C12 myoblast cells. Moreover, ucOC enhances myogenic differentiation via a mechanism involving GPRC6A-ERK1/2 signalling.

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Extracellular xenogeneic hemoglobin suppresses the capacity for C2C12 myoblast myogenic differentiation

Archives of Biological Sciences ◽

10.2298/abs200625032s ◽

2020 ◽

Vol 72 (3) ◽

pp. 379-391

Author(s):

Ana Stancic ◽

Ivana Drvenica ◽

Branko Bugarski ◽

Vesna Ilic ◽

Diana Bugarski

Keyword(s):

Cell Therapy ◽

Experimental Model ◽

Myogenic Differentiation ◽

Calf Serum ◽

C2c12 Cells ◽

C2c12 Myotubes ◽

Heme Oxygenase 1 ◽

C2c12 Myoblast ◽

Free Hemoglobin ◽

Functional Characteristics

Functional characteristics of satellite cells (SCs) that act as myogenesis initiators and have emerged as a promising target for cell therapy, are dependent on their microenvironment. The aim of this study was to investigate the effect of cell-free hemoglobin, as a part of the microenvironment of SCs, on their functional characteristics. The C2C12 cell line served as the experimental model of SCs; hemoglobin isolated from porcine (PHb) and bovine (BHb) slaughterhouse blood served as the experimental model for extracellular hemoglobin. The proliferation rate of C2C12 cells was assessed by the MTT test, migration capacity by the scratch assay, and myogenic differentiation capacity by histochemical staining and RT-PCR analysis of the expression of genes specific for myogenic lineage. The effect of hemoglobin on the proliferation and migration of C2C12 cells was dependent on its concentration and the animal species it was isolated from, but the effect of BHb was more prominent. Both PHb and BHb decreased the expression levels of myogenin and muscle specific creatine kinase at a 10 ?M concentration. While PHb had no effect on the morphometric parameters of C2C12 myotubes, BHb modified the area and length of C2C12 myotubes cultivated in DMEM/2% horse serum and DMEM/10% fetal calf serum. While PHb and BHb had no effect on heme oxygenase 1 (Hmox1) expression, they stimulated the expression of hypoxia-inducible factor 1-alpha (Hif1?) at a concentration of 10 ?M. The mainly inhibitory effect of cell-free hemoglobin on myogenic differentiation suggests that it could be a relevant factor in the outcome of cell therapy of muscle injury.

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Ganoderma microsporum immunomodulatory protein, GMI, promotes C2C12 myoblast differentiation in vitro via upregulation of Tid1 and STAT3 acetylation

PLoS ONE ◽

10.1371/journal.pone.0244791 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0244791

Author(s):

Wan-Huai Teo ◽

Jeng-Fan Lo ◽

Yu-Ning Fan ◽

Chih-Yang Huang ◽

Tung-Fu Huang

Keyword(s):

Myogenic Differentiation ◽

Atp Synthesis ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

C2c12 Myoblast ◽

Muscle Loss ◽

C2c12 Myoblasts ◽

Immunomodulatory Protein ◽

Pre Treatment

Ageing and chronic diseases lead to muscle loss and impair the regeneration of skeletal muscle. Thus, it’s crucial to seek for effective intervention to improve the muscle regeneration. Tid1, a mitochondrial co-chaperone, is important to maintain mitochondrial membrane potential and ATP synthesis. Previously, we demonstrated that mice with skeletal muscular specific Tid1 deficiency displayed muscular dystrophy and postnatal lethality. Tid1 can interact with STAT3 protein, which also plays an important role during myogenesis. In this study, we used GMI, immunomodulatory protein of Ganoderma microsporum, as an inducer in C2C12 myoblast differentiation. We observed that GMI pretreatment promoted the myogenic differentiation of C2C12 myoblasts. We also showed that the upregulation of mitochondria protein Tid1 with the GMI pre-treatment promoted myogenic differentiation ability of C2C12 cells. Strikingly, we observed the concomitant elevation of STAT3 acetylation (Ac-STAT3) during C2C12 myogenesis. Our study suggests that GMI promotes the myogenic differentiation through the activation of Tid1 and Ac-STAT3.

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Nogo-A is critical for pro-inflammatory gene regulation in myocytes and macrophages

10.21203/rs.3.rs-84136/v1 ◽

2020 ◽

Author(s):

H M Arif Ullah ◽

A. K. Elfadl ◽

SunYoung Park ◽

Yong Deuk Kim ◽

Myung-Jin Chung ◽

...

Keyword(s):

Inflammatory Process ◽

Vital Role ◽

C2c12 Cells ◽

Inflammatory Factors ◽

Mdx Mice ◽

C2c12 Myoblast ◽

Homologous Protein ◽

Inflammatory Processes ◽

Myoblast Cells ◽

Factor C

Abstract Background: Nogo-A (Rtn 4A), a member of the reticulon 4 (Rtn4) protein family, is a neurite outgrowth inhibitor protein that is primarily expressed in the central nervous system (CNS). However, the role of Nogo-A in inflammatory mechanisms remains unclear. Therefore, in this study, we used Nogo-knockout (KO) mice to explore its potential role in the inflammatory process. Here, we investigated whether Nogo-A affects the inflammatory process through transcription factor C/EBP homologous protein (CHOP). Results: Our results demonstrated that Nogo-A, CHOP, and pro-inflammatory factors were activated in the following: notexin-induced muscle injury, in human Duchenne muscular dystrophy (DMD) patients, in dystrophin-deficient (mdx) mice, in differentiated C2C12 myoblast cells, and in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDM). Moreover, we found that Nogo-KO BMDM exhibited lower migratory ability compared with wild type (WT) BMDM after LPS treatment. Conclusion: Our data demonstrated that the Nogo-A-CHOP signaling pathway regulated the inflammatory process in notexin-induced injured muscle, in mdx mice, in DMD patients, in differentiated C2C12 cells, and in LPS-stimulated BMDM. Taken together, these results suggest that Nogo-A plays a vital role in inflammatory processes, which resembles the pathological mechanisms observed in the CNS.

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Lipid droplets contribute myogenic differentiation in C2C12 by promoting the remodeling of the acstin-filament

Cell Death and Disease ◽

10.1038/s41419-021-04273-8 ◽

2021 ◽

Vol 12 (12) ◽

Author(s):

Yanjie Tan ◽

Yi Jin ◽

Pengxiang Zhao ◽

Jian Wu ◽

Zhuqing Ren

Keyword(s):

Skeletal Muscle ◽

Actin Filaments ◽

Lipid Droplets ◽

Myogenic Differentiation ◽

C2c12 Myoblast ◽

Cellular Processes ◽

Myoblast Cells ◽

Athlete’S Paradox ◽

The Impact ◽

Insight Into

AbstractLipid droplet (LD), a multi-functional organelle, is found in most eukaryotic cells. LDs participate in the regulation of many cellular processes including proliferation, stress, and apoptosis. Previous studies showed the athlete’s paradox that trained athletes accumulate LDs in their skeletal muscle. However, the impact of LDs on skeletal muscle and myogenesis is not clear. We discovered that C2C12 myoblast cells containing more LDs formed more multinucleated muscle fibers. We also discovered that LDs promoted cell migration and fusion by promoting actin-filaments remodeling. Mechanistically, two LD-proteins, Acyl-CoA synthetase long chain family member 3 (ACSL3) and lysophosphatidylcholine acyltransferase 1 (LPCAT1), medicated the recruitment of actinin proteins which contributed to actin-filaments formation on the surface of LDs. During remodeling, the actinin proteins on LDs surface translocated to actin-filaments via ARF1/COPI vesicles. Our study demonstrate LDs contribute to cell differentiation, which lead to new insight into the LD function.

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Biomechanical signals upregulate myogenic gene induction in the presence or absence of inflammation

AJP Cell Physiology ◽

10.1152/ajpcell.00594.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. C267-C276 ◽

Cited By ~ 35

Author(s):

Ravi Chandran ◽

Thomas J. Knobloch ◽

Mirela Anghelina ◽

Sudha Agarwal

Keyword(s):

Cell Differentiation ◽

Muscle Cell ◽

Kinase Inhibitor ◽

Cell Damage ◽

Myogenic Differentiation ◽

C2c12 Cell ◽

Gene Induction ◽

C2c12 Cells ◽

Proinflammatory Mediators ◽

Tnf Α

Inflammation of the muscle invariably leads to muscle cell damage and impaired regeneration. Biomechanical signals play a vital role in the regulation of myogenesis in healthy and inflamed muscle. We hypothesized that biomechanical signals counteract the actions of proinflammatory mediators and upregulate the basic helix-loop-helix and MADS box transcription enhancer factor 2 (MEF2) families of transcription factors, leading to increased myogenesis in inflamed muscle cells. For this purpose, C2C12 cells plated on collagenized silastic membranes were subjected to equibiaxial cyclic tensile strain (CTS) in the presence or absence of TNF-α, and the myogenic gene induction was examined over a period of 72 h. Exposure of cells to CTS resulted in a significant upregulation of mRNA expressions and synthesis of myogenic regulatory factors, MYOD1, myogenin (MYOG), MEF2A, and cyclin-dependent kinase inhibitor 1A (CDKN1A; p21) as well as muscle structural proteins like myosin heavy chain (MYHC) isoforms (MYH1, MYH2, and MYH4) and α-tropomyosin (TPM1), eventually leading to an increase in myotube formation. Contrarily, TNF-α suppressed the expression of all of the above differentiation-inducing factors in C2C12 cells. Further results revealed that simultaneous exposure of C2C12 cells to CTS and TNF-α abrogated the TNF-α-mediated downregulation of myogenic differentiation. In fact, the mRNA expression and protein synthesis of all myogenic factors ( Myod1, Myog, Mef2a, Cdkn1a, Myh1, Myh2, Myh4, and Tpm1) were increased in stretched C2C12 cells despite the sustained presence of TNF-α. These results demonstrate that mechanotransduction regulates multiple signaling molecules involved in C2C12 cell differentiation. On one hand, these signals are potent transducers of myotube phenotype in myoblasts; on the other, these signals counteract catabolic actions of proinflammatory cytokines like TNF-α and allow the expression of myogenic genes to upregulate muscle cell differentiation.

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p27Kip1 Acts Downstream of N-Cadherin-mediated Cell Adhesion to Promote Myogenesis beyond Cell Cycle Regulation

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0612 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1469-1480 ◽

Cited By ~ 37

Author(s):

Graziella Messina ◽

Cristiana Blasi ◽

Severina Anna La Rocca ◽

Monica Pompili ◽

Attilio Calconi ◽

...

Keyword(s):

Cell Cycle ◽

Myogenic Differentiation ◽

Cell Cycle Control ◽

C2c12 Cell ◽

Terminal Differentiation ◽

Active Role ◽

C2c12 Cells ◽

Low Density

It is widely acknowledged that cultured myoblasts can not differentiate at very low density. Here we analyzed the mechanism through which cell density influences myogenic differentiation in vitro. By comparing the behavior of C2C12 myoblasts at opposite cell densities, we found that, when cells are sparse, failure to undergo terminal differentiation is independent from cell cycle control and reflects the lack of p27Kip1 and MyoD in proliferating myoblasts. We show that inhibition of p27Kip1 expression impairs C2C12 cell differentiation at high density, while exogenous p27Kip1 allows low-density cultured C2C12 cells to enter the differentiative program by regulating MyoD levels in undifferentiated myoblasts. We also demonstrate that the early induction of p27Kip1 is a critical step of the N-cadherin-dependent signaling involved in myogenesis. Overall, our data support an active role of p27Kip1 in the decision of myoblasts to commit to terminal differentiation, distinct from the regulation of cell proliferation, and identify a pathway that, reasonably, operates in vivo during myogenesis and might be part of the phenomenon known as “community effect”.

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Fibroblast Growth Factor 21 Promotes C2C12 Cells Myogenic Differentiation by Enhancing Cell Cycle Exit

BioMed Research International ◽

10.1155/2017/1648715 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Xinyi Liu ◽

Yongliang Wang ◽

Shuhong Zhao ◽

Xinyun Li

Keyword(s):

Cell Cycle ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Myogenic Differentiation ◽

C2c12 Cell ◽

C2c12 Cells ◽

Fibroblast Growth Factor 21 ◽

Fibroblast Growth ◽

Cell Cycle Exit ◽

Protein Functions

Fibroblast growth factor 21 (FGF21), a secretion protein, functions as a pivotal regulator of energy metabolism and is being considered as a therapeutic candidate in metabolic syndromes. However, the roles of FGF21 in myogenic differentiation and cell cycle remain obscure. In this study, we investigated the function of FGF21 in myogenesis and cell cycle exit using C2C12 cell line. Our data showed that the expression of myogenic genes as well as cell cycle exit genes was increased after FGF21 overexpression, and FGF21 overexpression induces cell cycle arrest. Moreover, cell cycle genes were decreased in FGF21 overexpression cells while they were increased in FGF21 knockdown cells. Further, FGF21/P53/p21/Cyclin-CDK has been suggested as the key pathway for cell cycle exit mediated by FGF21 in C2C12 cells. Also, we deduce that FGF21 promotes the initiation of myogenic differentiation mainly through enhancing cell cycle exit of C2C12 cells. Taken together, our results demonstrated that FGF21 promotes cell cycle exit and enhances myogenic differentiation of C2C12 cells. This study provided new evidence that FGF21 promotes myogenic differentiation, which could be useful for better understanding the roles of FGF21 in myogenesis.

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